Embryonated chicken eggs still are one of the main substrates for the production of human vaccines. They are able to support the replication of a wide range of human and animal viruses. This spectrum includes attenuated viruses, i.e. defective viruses that have impaired potential to replicate in human or mammalian cells and can thus be used as vaccines. Attenuation can be generated or maintained by continuous passage in embryonated eggs. Chicken eggs used for human vaccine production must be certified to be free of a defined set of viral and bacterial contamination (specific pathogen-free or SPF). SPF eggs are available from commercial suppliers. The broad applicability and a long international track record has kept this strategy alive despite clear disadvantages:
SPF flocks of chicken and embryonated eggs are expensive and can constitute up to 40% of the cost of vaccines. Furthermore, it is difficult to continually maintain SPF flocks completely free of pathogens which is evidenced by periodic outbreaks of disease in SPF flocks. A vaccine lot cannot be released until the SPF supplier verifies that the parental chickens for the embryonated eggs used to manufacture the vaccine lot were completely free of any disease. This uncertainty adds a significant cost to the preparation of these vaccines. In pandemic situations with sudden need for a particular vaccine (e.g. influenza) the supply of SPF eggs may be severely limited. In addition, the large-scale processes for infecting eggs and maintaining virus growth are time consuming and sometimes inconsistent across different vaccine batches.
With the development of cell culture techniques vaccine manufacturers have replaced embryonated eggs with isolated chicken embryonic fibroblasts. While the use of primary cell cultures improves the safety profile, efficiency and reliability of the manufacturing process, it also further increases costs: chicken fibroblasts are prepared from SPF eggs by mincing embryos to establish and amplify viable cells. Typical for primary animal cells the fibroblasts suffer senescence: the doubling time increases with passaging and eventually all cells die. This process occurs after about 20 passages, much earlier than for rodent or some human cell substrates currently used in vaccine manufacture (such as MRC-5 or WI-38). Fibroblast cultures have to be maintained in the presence of 5-10% fetal calf serum, adding additional risk factors to the manufacturing process. They also require a solid surface for propagation and do not grow in suspension, a preferred state for bioreactor applications. Even with the use of multilayer cell factories this substantially limits scale-up procedures. Due to the limited live span a complete set of safety tests has to be applied for each lot of chicken fibroblasts.
Fibroblasts are the only cell type out of the wide variety of different tissues from a chicken embryo that proliferates well. The predominance of fibroblasts compared to other cell types has in some cases decreased theoretical virus yield because in eggs typically the chorioallantoic membrane, an epithelial cell layer, is the main site for virus amplification.
The discussed problems have contributed to severe influenza vaccine shortages in the last two years (2003 and 2004). To overcome these limitations, a permanent cell line growing in a synthetically defined medium, preferably in suspension or at least on carriers, would be highly desired.
Some of the viruses typically grown in chicken fibroblasts have been adapted to certain cell lines. BHK-21 (baby hamster kidney) cells support the growth of various vaccinia, influenza, and rabies vaccine strains (Drexler, I. et al., J. Gen. Virol. 79(Pt2):347-52 (1998); Gumusderelioglu M. et al., Biotechnol. Appl. Biochem. 33:167-72 (2001); Merten, O. W. et al., Adv. Exp. Med. Biol. 397:141-51 (1996)) and easily grow in large fermenters on carriers under serum-free conditions (Pay, T. W. et al., Dev. Biol. Stand 60:171-4 (1985); Gallegos Gallegos, R. M. et al., Arch. Med. Res. 26:59-63 (1995)). For vaccinia this applies even to the highly attenuated strain Ankara (MVA) which was developed on chicken cells. The BHK-21 cell line is accepted for production of certain vaccines for livestock animals (Lubiniecki, A. S., Bioprocess Technol. 10:495-513 (1990)). However, the BHK-21 line does not meet the safety requirements for human live vaccines. BHK cells have spontaneously formed, are highly tumorigenic and their history is inadequately reported.
According to the FDA, CBER Discussion from May 12 , 2000 on cell substrates the development of “Minimally-Purified Live-Attenuated Viral Vaccines and Virus-Vectored Vaccines” in neoplastic cells derived from naturally occurring tumors from humans and other mammals or from human cells and mammalian cells that have been transformed by unknown mechanisms is discouraged.
As an exception to the rule the VERO cell line (originating from African green monkey) is allowed as a cell substrate for vaccine manufacture based on a proven safety profile and the lack of transformed phenotype for a defined number of passages. The cell line has been used extensively for the manufacture of the polio and smallpox vaccines for clinical use. However, VERO cells require attachment and are amenable only to carrier based processes.
Additionally MDCK cells (a spontaneous cell line from dog kidney epithelium) with a described history have been applied to the manufacture of influenza virus (Tree, J. A. et al., Vaccine 19:3444-50 (2001)).
More recently, triggered by the development of vector based vaccines and gene therapy approaches, new so-called designer cell lines of human origin are intensely discussed and included into the spectrum of potential cell substrates for vaccine production (Vaccines and Related Biological Products advisory committee, session from May 16, 2001). New permanent cell lines were created to provide complementing genes for recombinant viruses that are replication-deficient outside the production system. However, stable introduction of the complementing genes requires prolonged cultivation times, which either exceed the natural limit of passage numbers available to primary cells or the tolerated limit of passage numbers for VERO cells before full transformation occurs.
Designer cell lines are generated in vitro with extensive documentation using characterized genes for transformation. For example, the complementing genes from the E1 region of adenoviruses by themselves exhibit transforming properties and have allowed establishment of human cell lines, for example PER.C6 (Fallaux, F. J. et al., Hum. Gene Ther. 9:1909-17 (1998)). The application of these cell lines is not limited to the viral vector they are designed for but may be extended to other viruses. For example, influenza virus can be propagated on PER.C6 (Pau, M. G. et al., Vaccine 19:2716-21 (2001)). However, this finding does not apply to all viruses relevant to vaccine development, in particular avian viruses such as Marek's disease, infectious bursal disease, Newcastle disease, turkey herpes, or chicken anemia viruses. While some of these viruses replicate well on mammalian cell lines, virus growth is often poor. For other viruses, replication is poor and limited to particular especially adapted strains.
In addition, with adaptation to a primate-derived cell substrate, receptor binding sites on the virus are likely to change resulting in a modified antigen pattern and thus a general effect on immunogenicity. This genetic adaptation may reverse attenuation for strains which have been developed via passaging in avian cells such as MVA or chicken-adapted measles virus (Escoffier, C., Gerlier, D., J. Virol. 73:5220-4 (1999)), or create new strains replicating more efficiently in human cells compared to their wild type isolates. Such viruses may also obtain a higher pathogenic potential.
For the above reasons vaccine manufacturers are reluctant to switch to mammalian cell lines and a need for immortal avian cell lines has developed.
The investigation of tumor induction in birds by the avian alpharetroviruses provided first molecular insights on cell transformation in general. The retroviral oncogenes are derived from cellular genes with essential regulator domains mutated or deleted. Some of the factors that have been identified in the course of these studies, such as v-myc or v-ras, directly affect components of both retinoblastoma (RB) and p53 pathways. Other proteins, such as v-src or v-erbB, are constitutively activated (hence, dysregulated) signal transducers that mimic impinging extracellular mitogens. The problem with these factors is that they target only one of several pathways required for efficient transformation. The presence of v-src or v-myc predisposes the cell for transformation and requires additional, spontaneous and unpredictable alterations within the cell for full transformation. The risks for the patient posed by cells transformed with one of the retroviral oncogenes therefore is difficult to estimate.
In other cases a single strong tumor antigen (e.g. v-jun) is able to directly cause tumor formation (Hartl, M. et al., Curr. Cancer Drug Targets 3:41-55 (2003)). Many avian viral oncogenes maintain their oncogenic potential in mammalian cells.
Cell lines created by these viruses are not suitable for vaccine manufacturing. A retrovirus carrying an oncogene may get activated and transferred together with the vaccine. Even a tumor antigen not enclosed by viral LTRs may pose a high risk when it is able to transform mammalian cells without the help of complementary antigens. This risk is typically estimated by consideration of the transforming potential, the number of vaccinees, and the amount of cellular nucleic acid transferred with the vaccine virus. This amount is limited by the efficiency of the purification process and currently cannot be reduced to below 10 pg/dose. This criterion is especially stringent for vaccine production where a healthy population often is inoculated at a very young age.
The same arguments apply to transforming DNA viruses such as papillomaviruses and polyomaviruses. These viruses are equiped with aggressive oncogenes: SV40 large T antigen is a multifunctional protein which affects both checkpoint control in G1 of the cell cycle and p53 activity. Therefore, large T readily immortalizes and transforms multiple mammalian tissues of rodent and human origin. With the addition of small T antigen (further enhancing large T action and additionally modulating the AKT3 pathway) it was possible to immortalize avian cells (part of patent application US 2001-0016348). However, even with sophisticated modern purification methods SV40 large-T antigen is considered too aggressive for use in cell lines generated for application in human medicine. In contrast to the above, the genes proposed in this invention affect checkpoint control of the cell cycle and p53 inactivation via separate factors: a required simultaneous transfer event of two distinct factors for transformation dramatically decreases any theoretical risk for the vaccinee.
US patent application 2001-0016348 describes the use of an anti-apoptotic pathway completely unrelated to the present invention. It does not provide a second gene that counters an internal signal for apoptosis due to forced cell cycle progression caused by a first gene. Apoptosis can also be induced by a variety of external simuli, for example lack of growth factors or loss of anchorage. Transmission of this type of pro-apoptotic signal can be inhibited by bcl-2 family genes, the focus of US patent application 2001-0016348.
Whereas 90% of cervix carcinomas carry papillomavirus sequences, C-type adenoviruses (which include types 2 and 5) are considered not to induce tumors in vivo, and adenoviral sequences have not been detected in human tumor tissue.
Alternatively, it has been tried to develop cell lines by continuous passaging of chicken embryonic fibroblasts. Whereas rodent cells appear to undergo spontaneous immortalization quite easily (Curatolo et al., In Vitro 20:597-601 (1984)), avian and primate cells are highly resistant to this approach (Harvey, et al., Genes and Development 5:2375-2385 (1991); Pereira-Smith, J. Cell Physiol. 144:546-9 (1990); Smith et al., Science 273:63-67 (1996)). Somatic cells of avian or primate origin lack telomerase and senescence is caused by the shortening of chromosomal ends (telomeres). Nevertheless, a chicken fibroblast line UMNSAH-DF1 has been developed using this approach (U.S. Pat. Nos. 5,672,485 and 6,207,415). Immortalization by this approach is caused by spontaneous mutations in multiple oncogenes or tumor suppressor genes. This is a rare event which is unlikely to be reproduced especially in cells of other tissue origin. Most importantly, such an approach contradicts the Defined Risk approach as a general rule for human live vaccines proposing detailed knowledge about the immortalizing genes to assess the risk of oncogene transfer. Again, according to the FDA (CBER Discussion from May 12, 2000, on cell substrates) the use of neoplastic cells derived from naturally occurring tumors or cells that have been transformed by unknown mechanisms is discouraged for the development of minimally-purified live-attenuated viral vaccines and virus-vectored vaccines.
The spontaneously developed UMNSAH-DF1 chicken fibroblast line exhibits alterations in E2F and p53 activity (Kim et al., Oncogene 20: 2671-82 (2001)). This is not surprising because enhanced cell cycle activity requires active E2F, and because it is known from mammalian cell studies that high E2F activity induces apoptosis in the presence of active p53. The study characterizes the immortal stage without shedding light on the causative events: mutations in a large number of genes may have caused immortalisation.
A spontaneous transformation process may be enhanced by the use of chemical mutagens (U.S. Pat. No. 5,989,805). The particular cell lines generated using this approach have overcome senescence but maintained a fibroblast like appearance and are non-tumorigenic. Although this represents a significant safety feature, these cells are of low value for large scale fermentation techniques. Furthermore, this chance-based approach also contradicts the Defined Risk guidelines.
Avian cell lines originating from naturally occurring tumors such as a quail fibrosarcoma (WO 97/08307) have also been proposed for biomanufacturing. Again, the Defined Risk guidelines for use in human vaccine production are violated by a method that is based on chance events.
The approaches taken in the studies described above are in sharp contrast to the active introduction of specific groups of immortalising genes according to this invention, which defines the causative agents for immortalisation and allows to assess risk, provides high flexibility with respect to selection of various tissues, and allows to modulate certain features of the resulting cell line.
Despite the fact that chicken eggs and fibroblasts have a considerable track record they are also associated with a very specific risk factor that only recently has come into greater focus: chicken cells release at least two types of retroviral particles, the endogenous avian retrovirus (EAV) and the endogenous avian leukosis virus (ALV-E). The issue is similar to the presence of endogenous retrovirus particles in mouse cells which are used for the manufacture of recombinant proteins (such as NSO). However, in contrast to mouse cells, chicken cells have been shown to contain reverse transcriptase. Due to more efficient detection techniques RT activity has also been detected in chicken cell-derived measles, mumps and yellow fever vaccines (Hussain, A. I. et al., J. Virol. 77:1105-11 (2003); Shahabuddin, M. et al., J. Clin. Microbiol. 39:675-84 (2001)). Whether the presence of reverse transcriptase activity results in transmissible retroviruses remains controversial: a more detailed analysis has shown that CEF (from White Leghorn) contain five loci with integrated EAVs, two of which can express infectious ALV-E whereas the other three are defective (Johnson, J. A., Heneine, W., J. Virol. 75:3605-12 (2001)). Tsang, S. X. et al., J. Virol. 73:5843-51 (1999) also found RT activity and release of viral particles but did not observe any transmission after a careful search for EAV sequences in blood mononuclear cells of children that received mumps vaccine. According to the Weekly Epidemiological Record of the WHO (73) 28 (1998), independent laboratories have investigated the infectivity of the particles for a variety of human and other mammalian cells by extensive co-cultivation and could not detect transmission of RT activity or productive infection. This finding is supported by epidemiological studies that have revealed no association between the use of chicken cell-derived vaccines and incidence of cancers, including those of childhood.
Furthermore, in the mentioned Weekly Epidemiological Record, the WHO stresses the importance of chicken host cells to maintain attenuation of certain vaccine strains. Alternative production processes are not currently available, and this lack of alternatives is an important reason for the acceptance of a known and continous source for a viral contaminant.
However, epidemiological studies superimpose populations and do not investigate chance events or case studies. Epidemiological studies cannot refute theoretical risks, for example: the accepted endogenous RT activity may mask RT activity from unacceptable exogenous contamination, and the endogenous viruses may be mobilized and activated if packaging constructs are introduced into the cells (Ronfort, C. et al., Virology 207:271-5 (1995)).
It was shown, however, that cells from ducks and geese do not contain EAV and ALV related sequence and the Japanese quail is free of reverse transcriptase (Smith, L. M. et al., J. Gen. Vrol. 80(pt1):261-8 (1999); Brudno, I. A. et al., Vopr. Virusol. 97-100 (1980)).
Adenoviruses (AdV) are well characterized, naked (non-enveloped) ubiquitous viruses. For the most common serotypes Ad2 and Ad5 the seroprevalence in the human population approaches 90%. Replication incompetent versions of these viruses are used as gene therapy and vaccine vectors in trials with human patients. Genes from the E1 region of human Adenovirus 5 have been used to transform some specific human cells in vitro (293 and PER.C6 cell lines; Fallaux, F. J. et al., Hum. Gene Ther. 9:1909-17 (1998); Graham, F. L. et al., J. Gen. Virol. 36:59-74 (1977)). The general process is inefficient compared to stronger multifunctional oncogenes such as SV40 large T antigen. Based on the observation that 293 show neuron specific markers and PER.C6 are of neuroectodermal origin it was suggested that Ad5 E1-based transformation is limited to neuronal cells (Shaw et al. Faseb J 16(8): 869-71(2002)). Considering the significant species barrier between human and avian cells efficient immortalisation of multiple avian tissues by transfection is even more unexpected.
Mammalian E1 transformed cell lines have been used for the production of live purified adenovirus vectors in clinical trials. With careful monitoring of the amount of contaminating cellular DNA in a vaccine preparation and its size, the transforming genes of Ad5 are not considered a safety hurdle (Vaccines and Related Biological Products advisory committee, session from May 16, 2001).
Adenoviruses replicate in the nucleus of the infected cell. Because quiescent host cells are not permissive for a full viral life cycle adenoviruses have evolved mechanism to force cells into S-phase. To maximize burst size of progeny viruses they have also evolved mechanism to evade apoptosis as a response of the host cell to capsid penetration and viral replication. The genomic region that mediates both cell cycle progression and inhibition of apoptosis is the E1 region.
The E1 region actually consists of two distinct expression cassettes, E1A and E1B, arranged in tandem and each equipped with its own promoter and polyadenylation site. At least three proteins are translated from the E1A primary transcript by alternative splicing. Among others, E1A proteins have been found to disrupt RB/E2F complexes and to interfere with the p300 and CBP transcriptional co-activators. The escape of E2Fs from the RB repressor induces progression of the cell cycle from G1 to S phase, whereas the E1A/p300 complex induces apoptosis via several pathways (Putzer, B. M. et al., Cell Death Differ. 7:177-88 (2000)), including repression of transcription of MdM2, a negative regulator of the key sensor for apoptosis, p53.
As E1A sensitizes cells to TNF-induced apoptosis it is considered an antitumor agent, and it is used in experimental approaches for tumor treatment (Lee, W. P. et al., Cancer Res. 63:6229-36 (2003)).
Furthermore, acting as a transcription modulator it drives cells towards de-differentiation, a feature advantageous to a potential cell substrate.
It was shown by Guilhot et al. (Guilhot, C. et al., Oncogene 8:619-24 (1993)) that retroviral transduction of the 12S protein of E1A from Ad5 can lead to immortalization of quail cells. This is likely the consequence of interaction between the avian RB and E1A. However, the process fails when the gene is introduced by transfection of naked DNA instead of retrovirus infection (pers. observation). We propose that the extremely efficient and stable transduction via retrovirus infection creates a cell pool large enough to harbor individual cells with spontaneous genomic changes that have blocked apoptosis that normally is induced upon RB inactivation. These required but unknown changes increase the risk for vaccinees and the resulting cell line cannot be considered a designer cell line (the result of defined blocks in specific pathways). Moreover, the transforming gene introduced via retroviruses is flanked by inverted terminal repeats and can, therefore, be mobilized. Such an event may even be more pronounced in cell lines that expresse reverse transcriptase from endogenous retroviruses.